Many naturally occurring proteins and peptides have been produced by recombinant DNA techniques. Recombinant DNA techniques have made possible the selection, amplification and manipulation of expression of the proteins and peptides. For example, changes in the sequence of the recombinantly produced proteins or peptides can be accomplished by altering the DNA sequence by techniques like site-directed or deletion mutagenesis.
However, some modifications to a recombinantly produced protein or peptide can not be accomplished by altering the DNA sequence. For example, the C-terminal .alpha.-carboxyl group in many naturally occurring protein and peptides often exists as an amide, but this amide typically is not produced through recombinant expressing and is biologically converted after expression in vivo from a precursor protein to the amide. Another example is the addition of a D-amino acid to the N- and/or C-terminal end of a recombinantly produced protein or peptide.
In addition, it may be desirable to selectively modify both the N- and C-terminal .alpha.-carbon reactive groups of a recombinantly produced protein or peptide. Recombinantly produced protein or polypeptides have a multiplicity of reactive side chain groups, as well as the N- and C-terminal amino acid .alpha.-carbon reactive groups. Side chain reactive groups include thiols, carboxyls, imidazoles, and .epsilon.-amine reactive groups. Selective modifications at the N- and/or C-terminal .alpha.-carbon reactive groups, such as adding an N-terminal pyroglutamyl residue and/or forming an amide at the C-terminal amino acid, need to be conducted without adversely affecting the reactive side chain groups.
A method of forming a C-terminal amide on a recombinantly produced polypeptide by the action of an enzyme is known. The enzyme is peptidyl glycine .alpha.-amidating monoxygenase and is present in eucaryotic systems. The enzyme has been used to form an amide on the C-terminal amino acid of recombinantly produced peptides, like human growth hormone releasing hormone in vitro as described by J. Engels, Protein Engineering, 1: 195-199 (1987).
In addition many recombinantly produced small proteins and peptides have a limited number of reactive side chain groups. For example, the 27 amino acid human gastrin releasing peptide contains N-terminal .alpha.-amine and side chain hydroxyl and .epsilon.-amine reactive groups. The myosin light chain kinase inhibitor contains 10 amino acids and has N-terminal .alpha.-amine and side chain .epsilon.-amine reactive groups. The C-terminal .alpha.-carboxyl groups are amidated in both of these naturally occurring peptides. Although these types of small proteins and peptides have a limited number of different reactive groups, they have been amidated through the traditional method of enzymatic C-terminal amidation. While selective, the enzymatic method is time consuming, expensive, gives unpredictable yields, and requires significant post reaction purification. The enzymatic method is also limited to modifying the recombinantly produced peptide by C-terminal amidation.
Accordingly, there is a need for a chemical method that provides for selective modification of either or both N-terminal .alpha.-amine and C-terminal .alpha.-carboxyl groups of a recombinantly produced polypeptide. This method results in selective modifications to one or both terminal amino acid .alpha.-carbon reactive groups and does not adversely affect the reactive side chain groups. There is also a need for a method of selective modification that allows addition of a variety of different organic moieties to the N- and/or C-terminal a-carbon reactive groups of a recombinantly produced polypeptide and that is convenient, cheap and capable of producing terminally modified recombinant polypeptides in high yield. Therefore, it is an object of the invention to develop a chemical method for selective modification of N-terminal .alpha.-amine and/or C-terminal .alpha.-carboxyl reactive groups of a recombinantly produced polypeptide.